Cystic fibrosis (CF) is the most common fatal recessive genetic disease among populations of European descent. It is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ion channel required for proper fluid and ion balance in multiple epithelial tissues. ~90% of CF patients carry one or two copies of the ?F508 allele, which causes inefficient protein folding, limited channel activity, and accelerated post-endocytic degradation. 'Modulator' compounds have been found that address the folding and channel defects. Neither provides benefit alone, but in combination they produce a modest, but statistically significant improvement in lung function (?FEV1 = 2.8%) in ?F508 homozygous patients. To reach more patients and increase the functional response, we are pursuing targets that specifically reduce the post-endocytic degradation of rescued ?F508-CFTR. Our work has identified the Disabled-2 (Dab2) protein as a key endocytic cargo adaptor for CFTR in human airway epithelial cells and validated an increase in both WT- and ?F508-CFTR levels following Dab2-specific RNA interference. We have also localized a target binding site by mutagenesis and shown that a peptide inhibitor of this site increases CFTR chloride currents. Thus, our central hypothesis is that Dab2 inhibitors will act in concert with existing CFTR modulators to boost rescue in the treatment of CF. To identify compounds that mimic the functionally validated peptide inhibitor of Dab2, our preliminary work has now engineered a high-affinity binding-site reporter, developed a fluorescence polarization (FP) assay with a robust signal-to-noise ratio in 384-well format (Z' = 0.76), and shown reproducible performance in a pilot high-throughput screen (HTS) at the Broad Outreach Laboratory. In parallel, we have established a full array of downstream assays, and have shown that two of our pilot hits can substantially enhance CFTR rescue over and above the VX-770/809 combination. In this application, we propose to combine our biochemical and cell-biological understanding of CFTR trafficking with the HTS and chemical biological expertise of the University of Pittsburgh Drug Discovery Institute (UPDDI) and the Gaslini Institute HTS lab to achieve the following specific aims: (1) Implement HTS of the NIH Molecular Libraries Small Molecule Repository (MLSMR: ~210,000 compounds) and a complementary UPDDI composite library (5,000 diverse compounds) and validate primary screening hits by FP at a distinct wavelength; (2) Rank these hits by cytotoxicity, potency, and CFTR half-life in combination with Vertex compounds in a cell-based assay, FP and FRET dose response, and counterscreen selectivity; (3) Characterize the stereochemistry and activity of candidates and related compounds, alone and in combination with CF modulators, using an array of tertiary and quaternary assays in cultured and then primary human airway epithelial cells. At the conclusion of these studies, we will have identified a new class of therapeutic candidates addressing the basic molecular defect of CF and provided a strong foundation for the mechanistic and pharmacological exploration of this novel target.